Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons
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Zusammenfassung
Growth cone intrinsic properties, such as intracellular cyclic nucleotide signalling pathways and ion channels in the growth cone plasma membrane, are important determinants of whether a guidance molecule functions as either an attractant or a repellent. Binding of guidance molecules to their receptor or receptor complexes triggers intracellular second messenger signalling cascades, e.g., via cAMP, cGMP and Ca2+, which ultimately leads to the regulation of appropriate cytoskeleton proteins in order to impose directional guidance on growth cones of either axons or dendrites.
Based on this background, this dissertation examined the mechanisms of second messenger signalling in response to diffusible guidance proteins, in particular the repellent Sema3A. Various techniques were utilized, including observations of single cell behaviour by growth cone turning assays combined with electrophysiology, immunocytochemistry, and Ca2+ imaging of cultured, developing Xenopus laevis spinal neurons. This work resulted in three specific conclusions: First, diffusible guidance molecules cause membrane potential shifts of about 15 mV; attractants cause depolarization and repellents cause hyperpolarization. Sema3A-induced cGMP production causes membrane hyperpolarization by the activation of Cl channels. In addition, pharmacological increase of cGMP levels leads to PKG-dependent activation of saxitoxin-sensitive channels, which converts Sema3A-induced repulsion to attraction. Thus, bi-directional turning responses are likely mediated by shifts of the membrane potential and the Sema3A-induced growth cone turning direction is imposed by the level of intracellular cGMP demonstrating a molecular switching mechanism of cGMP-dependent repulsion/hyperpolarization to PKG-dependent attraction/depolarization.
Second, Ca2+ influx through CNGCs is required and sufficient for Sema3A-induced growth cone repulsion. Electrophysiological studies indicated that CNG leak currents are enhanced in response to Sema3A. Xenopus spinal neuron growth cones express the CNGA1 subunit of rod-type CNGCs, which is co-expressed with the Sema3A receptor Npn-1. Loss-of-function studies, in which either antisense morpholino oligonucleotides against the Xenopus CNGA1 subunit were injected to eliminate the CNGA1 protein or a CNGA1 subunit mutant that has a deletion at the cGMP binding domain was overexpressed, abolish Sema3A-induced leak currents and convert the repulsion to attraction. Moreover, this conversion into attraction correlates with the conversion of membrane hyperpolarization to depolarization. Taken together, these observations indicate that Sema3A-induced bi-directional growth cone turning is caused by cGMP-dependent activation of CNGCs and likely VDCCs, depending on the level of intracellular cGMP. CNGCs or VDCCs in turn conduct Ca2+ in which the magnitude of Ca2+ entry determines the direction of growth cone turning.
Third, establishment and characterization of a culture system of dissociated primary mature Xenopus laevis spinal neurons allow the study of cellular and molecular mechanisms of nerve regeneration. This culture system reveals that older neurons display distinctive morphologies as well as different abilities to grow and survive than do their embryonic counterparts. In addition, consistent with the development in vivo, the polarity of their neurites in culture progressively switches from axons to dendrites as they are derived from increasingly mature developmental-stage animals. Importantly, unlike neurons in most other culture systems, these cultured neurons maintain their mature intrinsic properties. Interestingly, the culture conditions seem to favour the survival of commissural interneurons. Therefore, this culture system is a resource in which to study the mechanisms of nervous system function in mature animals and to examine the developmental changes that occur in the intrinsic properties of growth cones that may be responsible for the failure of adult nerve regeneration.
In summary, the three conclusions of this study contribute to our better understanding of the intracellular signalling mechanisms that regulate bidirectional growth cone turning in response to a diffusible guidance protein Sema3A, which functions not only during nervous system development, but also in various other important cellular processes, i.e., nerve regeneration, tissue differentiation and apoptosis. Finally, this study provides a pivotal research resource in form of a primary culture system for mature Xenopus spinal neurons that favours the survival of a distinct class of spinal neurons for the study of the cellular and molecular mechanisms of nerve regeneration.
Zusammenfassung in einer weiteren Sprache
Die intrinsischen Eigenschaften von Wachstumskegeln, wie z. B. intrazelluläre zyklische Nukleotidsignalwege und Ionenkanäle in der Plasmamembran, sind ausschlaggebend, ob ein Lenkungsmolekül entweder anziehend oder repulsiv wirkt. Binden Lenkungsmoleküle an ihren Rezeptor oder Rezeptorkomplex, wird eine intrazelluläre "second messenger" Signalkaskade, zum Beispiel über cAMP, cGMP oder Ca2+, ausgelöst, was letztendlich zur Regulation der entsprechenden Proteine des Cytoskelett führt und damit die Ausrichtung des Wachstumskegels einleitet.
Auf diesem Hintergrund untersucht diese Dissertation die Mechanismen der Signalübertragung durch second messenger infolge des diffusiblen Lenkungsmoleküls Sema3A. Hierzu werden verschiedene Verfahren verwendet, wie z. B. die Beobachtung des Einzelzellverhaltens durch "turning assays", in Kombination mit Elektrophysiologie, Immunfärbungen und Calcium-"Imaging" von kultivierten, sich entwickelnden Xenopus laevis Spinalneuronen. Die durchgeführten Versuche liefern drei wesentliche Erkenntnisse. Erstens verursachen diffusible Lenkungsmoleküle eine Verschiebung des Membranpotentials von etwa 15 mV, wobei anziehende Lenkungsmoleküle eine Depolarisation und repulsive Lenkungsmoleküle eine Hyperpolarisation herbeiführen. Hierbei verursacht die Sema3A-induzierte Produktion von cGMP eine Membranhyperpolarisation durch die Aktivierung von Chlorid-Kanälen. Außerdem führt eine pharmakologische Erhöhung des cGMP-Gehalts zu einer PKG-abhängigen Aktivierung von Natrium-Kanälen, was die Sema3A-induzierte Abstoßung in Anziehung umwandelt. Demzufolge werden bi-direktionale Wachstumsrichtungen durch Verschiebungen des Membranpotentials vermittelt und die Sema3A-induzierte Ausrichtung von Wachstumskegeln durch den intrazellulären cGMP-Gehalt bestimmt. Dies veranschaulicht einen molekularen Umschaltmechanismus, der die cGMP-abhängige Abstoßung/Hyperpolarisation in PKG-abhängige Anziehung/Depolarisation umwandelt.
Zweitens ist der Ca2+-Einstrom durch CNGCs sowohl notwendig als auch ausreichend für die Sema3A-induzierte Abstoßung von Wachstumskegeln. Elektrophysiologische Untersuchungen deuten darauf hin, dass CNG-Kriechstrom durch Sema3A erhöht wird. Desweiteren exprimieren Wachstumskegel von Xenopus Spinalneuronen die CNGA1-Untereinheit von Stäbchen-ähnlichen CNGCs, welche mit dem Sema3A-Rezeptor Npn-1 co-exprimiert ist. Untersuchungen zum Verlust der CNGC-Funktion, in denen entweder Antisense-Morpholino-Oligonukleotide gegen die CNGA1-Untereinheit von Xenopus injiziert wurden, um so das CNGA1-Protein auszuschalten, oder in denen eine CNGA1-Untereinheitmutante mit einer zerstörten cGMP-Bindungsdomäne überexprimiert wurde, zeigen die Beseitigung des Sema3A-induzierten Kriechstroms und eine Umwandlung der Abstoßung in Anziehung. Zudem korreliert diese Umwandlung mit der Umwandlung von Hyperpolarisation in Depolarisation. Zusammenfassend weisen diese Beobachtungen darauf hin, dass die Sema3A-induzierte bi-direktionale Ausrichtung des Wachstumskegels durch die cGMP-abhängige Aktivierung von CNGCs oder VDCCs verursacht wird, je nach intrazellulärem cGMP-Gehalt. CNGCs und VDCCs wiederum leiten Ca2+, wobei das Ausmaß des Ca2+-Einstroms die Ausrichtung des Wachstumskegels bestimmt.
Drittens ermöglicht die Entwicklung eines in vitro Kultursystems für dissoziierte, primäre, vollentwickelte Xenopus laevis Spinalneuronen, zelluläre und molekulare Mechanismen der Nervenregeneration zu untersuchen. Hierbei zeigt dieses Kultursystem, dass ältere Neuronen im Gegensatz zu ihren embryonalen Pendants sowohl unverkennbare Morphologien als auch unterschiedliche Wachstums- und Überlebensfähigkeiten haben. Zudem verschiebt sich die Polarität der Neuriten von Axonen zu Dendriten, wenn diese von zunehmend weiterentwickelten Tieren stammen, was mit der in vivo-Entwicklung übereinstimmt. Interessanterweise scheinen in diesen Kulturbedingungen vermehrt Kommissuralneurone zu überleben. Folglich ist dieses Kultursystem ein Hilfsmittel, mit dem die Funktionsmechanismen des Nervensystems in vollentwickelten Tieren und die entwicklungsabhängigen intrinsischen Wachstumskegeleigenschaften, die für das Scheitern der Regeneration adulter Nervenfasern verantwortlich sein könnten, untersucht werden können.
Zusammenfassend erweitern diese Erkenntnisse unser Wissen über die intrazellulären Mechanismen der Signalübertragung, welche die bi-direktionale Ausrichtung der Wachstumskegel infolge des diffusiblen Lenkungsmoleküls Sema3A regulieren, wobei Sema3A nicht nur während der Entwicklung des Nervensystems, sondern auch in anderen Zellprozessen, u.a. Nervenregeneration, Gewebeentwicklung und Apoptose, eine zentrale Rolle spielt. Schlussendlich bietet diese Studie mit der Entwicklung eines Kultursystems für vollentwickelte Xenopus Spinalneuronen ein zentrales Forschungshilfmittel, in dem eine bestimmte Spinalneuronenklasse überlebt, mit der die zellulären und molekularen Mechanismen der Nervenregeneration untersucht werden können.
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SCHIMMELMANN, Melanie Joan von, 2009. Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons [Dissertation]. Konstanz: University of KonstanzBibTex
@phdthesis{Schimmelmann2009Chann-8656, year={2009}, title={Ion Channel Regulation in Growth Cone Guidance by Semaphorin 3A in Xenopus laevis Spinal Neurons}, author={Schimmelmann, Melanie Joan von}, address={Konstanz}, school={Universität Konstanz} }
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Binding of guidance molecules to their receptor or receptor complexes triggers intracellular second messenger signalling cascades, e.g., via cAMP, cGMP and Ca2+, which ultimately leads to the regulation of appropriate cytoskeleton proteins in order to impose directional guidance on growth cones of either axons or dendrites.<br />Based on this background, this dissertation examined the mechanisms of second messenger signalling in response to diffusible guidance proteins, in particular the repellent Sema3A. Various techniques were utilized, including observations of single cell behaviour by growth cone turning assays combined with electrophysiology, immunocytochemistry, and Ca2+ imaging of cultured, developing Xenopus laevis spinal neurons. This work resulted in three specific conclusions: First, diffusible guidance molecules cause membrane potential shifts of about 15 mV; attractants cause depolarization and repellents cause hyperpolarization. Sema3A-induced cGMP production causes membrane hyperpolarization by the activation of Cl channels. In addition, pharmacological increase of cGMP levels leads to PKG-dependent activation of saxitoxin-sensitive channels, which converts Sema3A-induced repulsion to attraction. Thus, bi-directional turning responses are likely mediated by shifts of the membrane potential and the Sema3A-induced growth cone turning direction is imposed by the level of intracellular cGMP demonstrating a molecular switching mechanism of cGMP-dependent repulsion/hyperpolarization to PKG-dependent attraction/depolarization.<br />Second, Ca2+ influx through CNGCs is required and sufficient for Sema3A-induced growth cone repulsion. Electrophysiological studies indicated that CNG leak currents are enhanced in response to Sema3A. Xenopus spinal neuron growth cones express the CNGA1 subunit of rod-type CNGCs, which is co-expressed with the Sema3A receptor Npn-1. Loss-of-function studies, in which either antisense morpholino oligonucleotides against the Xenopus CNGA1 subunit were injected to eliminate the CNGA1 protein or a CNGA1 subunit mutant that has a deletion at the cGMP binding domain was overexpressed, abolish Sema3A-induced leak currents and convert the repulsion to attraction. Moreover, this conversion into attraction correlates with the conversion of membrane hyperpolarization to depolarization. Taken together, these observations indicate that Sema3A-induced bi-directional growth cone turning is caused by cGMP-dependent activation of CNGCs and likely VDCCs, depending on the level of intracellular cGMP. CNGCs or VDCCs in turn conduct Ca2+ in which the magnitude of Ca2+ entry determines the direction of growth cone turning.<br />Third, establishment and characterization of a culture system of dissociated primary mature Xenopus laevis spinal neurons allow the study of cellular and molecular mechanisms of nerve regeneration. This culture system reveals that older neurons display distinctive morphologies as well as different abilities to grow and survive than do their embryonic counterparts. In addition, consistent with the development in vivo, the polarity of their neurites in culture progressively switches from axons to dendrites as they are derived from increasingly mature developmental-stage animals. Importantly, unlike neurons in most other culture systems, these cultured neurons maintain their mature intrinsic properties. Interestingly, the culture conditions seem to favour the survival of commissural interneurons. Therefore, this culture system is a resource in which to study the mechanisms of nervous system function in mature animals and to examine the developmental changes that occur in the intrinsic properties of growth cones that may be responsible for the failure of adult nerve regeneration.<br />In summary, the three conclusions of this study contribute to our better understanding of the intracellular signalling mechanisms that regulate bidirectional growth cone turning in response to a diffusible guidance protein Sema3A, which functions not only during nervous system development, but also in various other important cellular processes, i.e., nerve regeneration, tissue differentiation and apoptosis. 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